Alkylation of DNA presents an important event of far reaching biological consequences. DNA alkylating agents are present endogenously, found in the environment, and are used in chemotherapy. Human beings are constantly exposed to alkylating agents from a wide variety of sources. For a better understanding of the changes at gene level responsible for DNA damage and a timely intervention in the DNA damaging process, availability of critical nucleosides, DNA and RNA to study the mechanism of DNA damage and repair is required. The information thus generated would have vast implication on understanding of various diseases that are the direct result of DNA damage from alkylation. Since all the alkylation and resulting mutation involve changes at gene level, the control of such damages at the gene level is an ideal approach.
Efforts are needed towards identification of alkylating agents, development of nucleosides and oligonucleotides which are suitable to synthesize alkylated DNA and RNA oligonucleotides, valuable in the study of optimum structural parameters for the reversal of cytotoxic DNA and RNA produced endogenously. This is essential, since application of the reversal process at early stages when mutation is at the beginning stage will help effective control of lesions that are at benign stages of toxic development.
The two main places of alkylation of DNA are O-alkylation and N-alkylation. Alkylation of DNA may lead to cytotoxic and mutagenic damage of the gene and gene products. However, the extent of damage depends upon the site of alkylation as well as on the nature of the base. N-Methylation of guanine occurs mainly at N7 position. However, this alkylation does not interfere with its pairing with cytosine and is therefore, harmless. The glycosidic bond of N7-methyl G undergoes slow and spontaneous hydrolysis creating a apurinic site which is a target for repair. On the other hand, N7-alkylation of G with a bifunctional agent such as nitrogen mustard causes cross-linking of two neighboring guanines leading to cell toxicity.
In the mono alkylation of adenine, the alkyl group at N3 occupies minor grove of the DNA double helix causing interference to DNA polymerase activity thus resulting in a major toxic lesion.
3-Alkyl guanines behave in a similar fashion as 3-alkyl adenines. However, formation of 3-alkyl guanines is much less prevalent (˜10 fold less) and therefore, of lesser significance. N1-alkylation of adenine interferes with A-T pairing. However, this lesion gets slowly excised in vivo.
O-Alkylation of the DNA bass fixes them in their enolic forms that may influence their base pairing. Thus, O6-methyl guanine forms an O6 Me G-C base pair that is more stable in a DNA duplex than O6-Me G-T base mispair. However, DNA polymerase activity shows preference for incorporation of Ton replication. The enolic forms of O4-methyl thymine and O4-ethyl thymine form base mispair with G that undergoes replication of a defined DNA sequence. However, alkyl pyrimidines are very minor products of DNA alkylation and are therefore, of low significance towards biological effects.
O-Alkylation of the phosphate residue in DNA results in the formation of a triester. Such triesters are repairable and do not seem to be important in inducing cell toxicity or mutagenic activity.
Therefore, from the above discussion it is clear that alkylation of DNA may cause cytotoxic and mutagenic damage depending upon the site of alkylation. This may have serious consequences in the formation of Gene and Gene products. In nature human beings are constantly exposed to alkylating agents from a wide variety of sources as mentioned above. Repair of the damaged DNA is therefore essential for normal activity. All living cells possess various DNA repair enzymes. These are more predominant in humans as compared to rodents. Under the influence of alkylating agents, E. coli respond by inducing the expression of four genes, ada, alkA, aidB, and alkB. The ada protein is an O6-methyl guanine-DNA methyltransferase and also regulates this adaptive response. AlkA is a 3-methyl adenine-DNA glycosylase, and aidB is meant to destroy certain alkylating agents. AlkB was isolated in 1983 by Kataoka et. al (Kataoka, H, Yamamoto, Y, & Sekiguchi, M., J. Bacteriol. 153, 1301-1307, 1983) but its exact role is still not clear. It has been shown to protect human cells against alkylation induced toxicity (Chen, B. J., Carroll, P., & Sanson, L., J. Bacteriol 176, 6255-6261, 1994). It processes the cytotoxic DNA damage generated in single-stranded DNA by SN2 methylating agents such as MeI, dimethylsulphate, methyl methanesulphonate etc. (Dinglay, S., Trewick, S. C., Lindahl, T., & Sedgwick, B., Genes Dev 14, 2097-2105, 2000). Its role in oxidative demethylation identified recently, is discussed under direct reversal of alkylated DNA. In a recent study it has been demonstrated that AlkB suppresses both genotoxicity and Mutagenesis at low doses of 1-alkylpurine and 3-alkylpyrimidine DNA damages in vivo (J. C. Delany and J. M. Essigmann, PNAS, USA, 101, 39, 14051-14056, 2004). Similarly it has been shown that N3-methylthymidine containing oligonucleotides could be repaired by AlkB in vitro (P. Koivisto, P. Robins, T. Lindahl, B. Sidgwick, J. Biol. Chem., 10, 1074, 2004).
There are two types of DNA repair mechanisms that operate in living cells: i) excision repair of altered residues and ii) direct reversal of modified DNA.
Excision repair is the most common mechanism that operates in mammalian cells for DNA repair. There are two types of excision repair: a) nucleotide excision and b) base excision. Both are error free mechanisms. In the nucleotide excision repair, an endonuclease initiates the process by making an excision on the single strand on either side of the lesion of the damaged nucleotide some 12 base apart. The excised nucleotide containing the lesion is released by DNA helicase B while it is still bound to the protein complex. The gap thus created on the single strand is filled up by DNA polymerase I that binds and fills the gap and subsequently the ligation is completed.
In base excision, the enzyme DNA glycosylase hydrolyses the glycosidic bond of modified purine or pyrimidine nucleosides. Thus, the enzyme 3-methyl adenine-DNA glycosylase acts on 3-methyl adenine. The next step is incision of the phosphate backbone by an AP endonuclease followed by exonuclease action which excises the nucleoside creating a gap on the single strand. Filling of the gap by DNA polymerase I followed by ligation as mentioned for nucleotide excision process, completes the repair process.
Direct reversal of damaged DNA also occurs under the influence of certain enzymes. Thus, O-demethylation in O6-methyl guanine is effected by the enzyme O6-methylguanine DNA methyltransferase. The SH group of Pro-Cys-His sequence of the enzyme near its C terminus, acts as a methyl acceptor, thus converting O-Me to OH. The same enzyme can also dealkylate ethyl, 2-hydroxyethyl, and 2-chloroethyl O6-derivatives of guanine

N-dealkylation is effected by alkB protein. Recently it has been shown by two independent groups (Faines, P. O., Johanson, R. F., & Seeberg, E., Nature, 419, 178, 2002; Trewick, S. C., Henshaw, T. F., Hausinger, R. P., Lindahl, T., & Sedgwick, B., Nature, 419, 174-178, 2002) that alkB resembles the Fe(ll)- and α-ketoglutarate-dependent dioxygenase. The enzyme couples oxidative decarboxylation of α-ketoglutarate to the hydroxylation of methylated bases in DNA, resulting in the reversal to unmodified base and release of formaldehyde as shown below:

Modified nucleosides and nucleotides find application in various chemical and biological studies. Such modifications have been linked to control of gene expression at both the levels of transcription and translation. For these studies, purified preparations of alkylated nucleosides and nucleotides are required. A large number of N-methyl purines have been isolated from various biological sources and identified (Jones, J. W., Robins, R. K., J. Am. Chem. Soc, 84, 1914, 1962).
Alkylation of various unprotected derivatives of guanine led to a mixture of products. alkylated at N1, N, N7, or O6 positions (Kamimura, T., Tsuchiya, M., Urakani, K. l., Kaura, K., Sekine, M., J. Am. Chem. Soc. 106, 4552-4557, 1984).
Extensive work has also been reported on synthetic approach to alkylated nucleosides and nucleotides. Bredreck and Martini (Bredereck, H., and Martini, A., Ber, 80, 401, 1948) treated triacetyl guanosine with excess diazomethane to obtain 1-methyl guanosine. However, the product was later shown to be 7-methyl guanosine (Jones, J. W., and Robins, R. K., J. Am. Chem. Soc. 85, 193-201, 1963). Methylation at pH 13-14 with dimethylsulphate in the presence of alkali gave a mixture of 7-methyl guanosine with methylation occurring at ribose unit as well. O6-Alkylation of guanosine has also been reported (Ramaswamy, K. S., Bakir, F., Baker, B., Cook, P. D., J. Heterocycl. Chem. 30, 1373-1378, 1993). Regioselective alkylation at N′ position of guanosine was achieved by Vincent et. al (Vincent, S. P., Mioskowski, C., and Lebeau, L., Nucleosides and Nucleotides, 18, 2127-2139, 1999).
Methylation of adenosine, 2′-deoxyadenosine, 2′-deoxyguanosine, inosine and xanthosine have been reported by Jones and Robins (Jones, J. W., and Robins, R. K., J. Am. Chem. Soc. 85, 193-201, 1963).
It has been found that the above mentioned alkylated derivatives undergo facile transformation such as Dimroth rearrangement, in which the methyl group is transferred from one nitrogen to a neighboring one. Thus, it has been reported that N′-methyl-2′-deoxyadenosine undergoes conversion into N6-methyl-2′-deoxyadenosine in 25% aq. ammonia with a half life of 36 hrs. In another study it was found that N1-methyl adenosine was stable for 3 days in 2M methanolic ammonia at room temperature and in 6 days time was totally converted into N6-methyl adenosine (Mikhailov, S. N., Rozensski, J., Efimtseva, E. V., Busson, R., Aershot, A. V., and Herdewijn, P., Nucleic Acid Res 30, 1124-1131, 2002; Maca, J. B., and Wolfender R., Biochemistry, 38, 13338-13346, 1968; Jones, J. W. and Robins, R. K., J. Am. Chem Soc, 85, 193-201, 1963). We found substantial migration of methyl group from N1 to N6 in the presence of 2M methanolic ammonia.
This instability of the molecule prevents their use for development of alkylated nucleosides and nucleotides.
Incorporation of N1-methyl adenosine into synthetic RNA oligonucleotides has been recently carried out in which the base moiety of N1-methyl adenosine was protected with chloroacetyl group at N6 position (Mikhailov, S. N., Rozensski, J., Efimtseva, E. V., Busson, R., Aerschot, A. V., and Herdewijn, P., Nucleic Acid Res 30, 1124-1131, 2002); subsequently, use of the same protecting group i.e. chloroacetyl, was reported for the preparation of N6-chloroacetyl-N1-methyl-riboadenosine by the same group (Efimtseva, E. V., Mikhailov S. N., Rozenski, J., Busson, van Aerschot, R. A. and Herdewijn., P. Poster No. P-156, 15th International Round Table Conference, Nucleosides, Nucleotides and Nucleic Acids, 10-14 September, 2002, Leuven, Belgium.
Efimtseva et al. also reported in the above mentioned Conference that the presence of N1-methyl adenosine in RNA destabilizes a duplex of RNA.
In order to study the effect of enzymes on dealkylation, it is important to synthesize well characterized alkylated nucleosides and nucleotides. Further, it has been observed that the introduction of a modified chloroacetyl protected base improves stability of a hairpin loop in RNA. Since these are important biochemical properties and bear strong implication of structure, function studies, it is important to ensure that correct and desired DNA and RNA are synthesized which are free from impurities, the side reaction products. Thus, development of nucleosides with specific protecting groups that prevent migration of the alkyl substituent in nucleosides during synthesis of desired oligonucleotides and oligodeoxynucleotides, has been carried out which forms the basis of the present invention.
Prior to utilize the N6 chloroacetyl protected N-1methyl-2′-deoxyadenosine 3′-cyanoethyl phosphoramidite, we carried out a number of kinetic studies for the deprotection of chloroacetyl group and determination of presence of any N-6 methyl-2′-deoxy adenosine, Dimroth rearrangement product, and surprisingly we observed N-6 methyl-2′-deoxy adenosine formation to the extent of 8-10%. Our subsequent studies to develop a ideal group which would be cleanly removed under aq. ammonia deprotection condition and would have negligible amount of Dimroth rearrangement product as bye product. N-6 FMOC N-1 merthyl-2′-deoxy adenosine-5′-DMT-3′-cyanoethylphosphoramidite emerged as best reagent with aq. ammonia deprotection taking place very cleanly, and the presence of N-6 methyl-2′-deoxy adenosine was found to be less than 1%.